Among many pathogenic microorganisms, the one casing trouble most over the world is Staphylococcus aureus, which is a Gram-positive coccus detected in approximately 30% of the total earth population and is known as a pathogen causing nosocomial infection (Isabel C. V, Robert S. K., Francisco A. K., J. Am. Acad. Dermatol. 50:845-849, 2004). This bacterium causes infection primarily on skin and soft tissue, leading to pyoderma, and further induces systemic infection threatening a life such as osteomyelitis, endocarditis, sepsis and bacteremia (Henry Shinefield, M. D. et al., The New England J. of Med. 346:491-496, 2002). Antibacterial agents for Staphylococcus aureus such as penicillin and methicillin have been used as a therapeutic agent for the infection over decades owing to their excellent antibacterial effect. However, since the strain exhibiting methicillin resistance (methicillin-resistant Staphylococcus aureus: MRSA) was first reported in England in 1961, the number of such antibiotics-resistant strains has been increasing (Standing Medical Advisory Committee, Department of Health, 1998).
The appearance of antibiotics resistant strains resulted from reckless overuse of antibiotics is also a serious problem in Korea. For example, the population of methicillin resistant strains reaches around 50% and according to the recent investigation by Korean Society for Nosocomial Infection Control, the rate of MRSA strains separated from most university hospitals and general hospitals reaches 70-80%. Therefore, it is required to develop a sanitary, safe and effective therapeutic agent for MRSA.
Korean Patent Application No. 2004-55648 describes that Streptomyces laidlogenes DS684 isolated from soil has the inhibitory effect on methicillin and vancomycin resistant strains, and Korean Patent No. 654370 describes that Lactobacillus ruminus SPM0211 has the inhibitory effect on vancomycin resistant strains.
Probiotics indicate the microorganism balancing intestinal microflora, the microorganism having antibacterial and enzyme activities and the product produced by the same (Fuller, R. J Appl Bacteriol. 66(5):365-378, 1989). Probiotics are also defined as live bacteria in the form of single strain or combined strains which can be supplied to human or animals in the form of dried cells or fermented products to improve intestinal microflora. Therefore, the probiotics use human intestines as a habitat and need to survive until they reach intestines as non-pathogenic and non-toxic. Further, the probiotics have to maintain the survival rate and activity in the product carrying the probiotics until they are consumed and have to be sensitive to antibiotics used for the prevention of infection and at the same time must not contain antibiotics resistant plasmid. Besides, the probiotics have to have resistance against acid, enzyme and bile in intestines (Mishra, C. et al., Asia Pacific J Clin Nutr. 5:20-24, 1996).
Probiotics are exemplified by Bacillus sp. having excellent digestive enzyme (amylase, protease, lipase, cellulose and phosphatase) producing capacity, Lactobacillus sp. capable of decomposing low molecular carbohydrates by producing organic acid, Saccharomyces sp. facilitating absorption in intestine after converting into lactic acid and vitamin B synthesis and vitamin E absorption as well, and Photosynthetic bacteria suppressing foul smell by metabolizing malodorous substances (ammonia, hydrogen sulfide, amines, etc.) remaining in feces of cattle.
In particular, Bacillus sp. and Lactobacillus sp. are generally called lactic acid bacteria (LAB) and separated from food. These species include strains that produce antibacterial substances working against saprogenic bacteria to prevent food from being rotten, which makes them very valuable probiotics (Tagg, G. U. et al., Bacteriol. Rev. 40:772-756, 1976). These lactic acid bacteria produce antibacterial peptide, so called bacteriocin, having antibacterial mechanism which has nothing to do with antibiotics resistance mechanism. Bacteriocin is not like the conventional antibiotics and is difficult to be defined precisely, since bacteriocin is not typical in molecular weight, biochemical characteristics, antibacterial spectrum to host or mechanisms. Klaenhammer defined bacteriocin as protein or protein complex that has antibacterial activity direct to those species close to bacteriocin producing bacteria (Klaenhammer, T R. Biochimie 70:337-379, 1998). The substances having antibacterial activity are suggested as “bacteriocin-like substances” even though they are not the typical ones identified according to the standard (Tagg, G. U. et al., Bacteriol. Rev. 40:772-756, 1976). There is no boundary fixed among antibiotics, bacteriocins and microcins, but bacteriocins are classified into bacteriostatics or bacteriocidals according to their activity spectrum and belong to peptide antibiotics group. Antibiotics are produced by non-ribosomal synthesis based on several steps of enzymatic mechanism. In the meantime, bacteriocins are generated by ribosomal synthesis and rarely have resistance but seem to have potential for resistance (Hurst A. Adv Appl Microbiol 27:85-123, 1981; Harvis, B., Farr, J. Biochim Biophys Acta 227:232-340, 1971; Harris, L J. et al., J Food Prot 52:384-387, 1989). Lots of antibiotics can be chemically synthesized but no chemical synthesis of bacteriocins has been reported, yet. With the advancement of genetic engineering techniques, bacteriocin-like substances can be reconstructed and recombinated, precisely lipids or carbohydrates take a part of bacteriocin complex. Bacteriocin can be adhered on cells or released out of the cells, can be generated before and after growth cycle, has resistance against such enzymes as protease, and is stable against pH and temperature. Antibacterial mechanism of bacteriocin has not been disclosed, yet, and only thing that has been reported is that bacteriocin is stuck in cell membrane channel to induce cell lysis. Cell lysis is directly associated with cell death and depends on sensitive bacteria having non-specific receptor site and specific receptor site (Bhunia, A K. et al., J Appl Bacteriol 57:492-498, 1991). Bacteriocin with these characteristics is rising as alternative antibiotics and is acknowledged as a safe antibacterial agent used as a food preservative.
Bacillus is a very interesting and industrially important species applicable in various industries, particularly regarding antibacterial activity, because it can produce numbers of peptide antibiotics and is safe for food and industrial use (Paik, H. D. et al., J. Ind. Microbiol. Biotechnol. 19:294-298, 1997). It has been known that many Bacillus species such as Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis produce bacteriocins and bacteriocin-like substances (Paik, H. D. et al., J. Ind. Microbiol. Biotechnol. 19:294-298, 1997; Zheng, G. et al., J. Bacteriol. 181:7346-7355, 1999; Bizani, D., Brandelli, A. J. Appl. Microbiol. 93:512-519, 2002).
The present inventors separated the novel Bacillus amyloliquefaciens K317 strain (KCTC 11042BP) from Kimchi, the Korean traditional safe fermented food, and further completed this invention by confirming that the culture solution of the strain and the antibacterial metabolite separated therefrom had strong inhibitory effect on various pathogenic enterobacteria including 30 kinds of MRSA strains.